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A double octarepeat prion protein construct, which has two histidines, mixed with copper sulfate in a 3:2 molar ratio provides at most three imidazole ligands to each copper ion to form a square-planar Cu2+ complex. This work is concerned with identification of the fourth ligand. A new (to our knowledge) electron paramagnetic resonance method based on analysis of the intense features of the electron paramagnetic resonance spectrum in the g⊥ region at 2 GHz is introduced to distinguish between three and four nitrogen ligands. The methodology was established by studies of a model system consisting of histidine imidazole ligation to Cu2+. In this spectral region at 2 GHz (S-band), g-strain and broadening from the possible rhombic character of the Zeeman interaction are small. The most intense line is identified with the MI = +1/2 extra absorption peak. Spectral simulation demonstrated that this peak is insensitive to cupric Ax and Ay hyperfine interaction. The spectral region to the high-field side of this peak is uncluttered and suitable for analysis of nitrogen superhyperfine couplings to determine the number of nitrogens. The spectral region to the low-field side of the intense extra absorption peak in the g⊥ part of the spectrum is sensitive to the rhombic distortion parameters Ax and Ay. Application of the method to the prion protein system indicates that two species are present and that the dominant species contains four nitrogen ligands. A new loop-gap microwave resonator is described that contains ∼1 mL of frozen sample.

Relaxation times have been obtained with time-domain EPR for the dinuclear mixed valence [CuA(1.5) ... CuA(1.5)[ S = 1/2 center in nitrous oxide reductase, N2OR, from Pseudomonas stutzeri, in the TN5 mutant defective in copper chromophore biosynthesis, in a synthetic mixed valence complex, and in type 1 and 2 copper complexes. Data confirmed that the intrinsic electron spin-lattice relaxation time, T1, for N2OR in the temperature range of 6-25 K is unusually short for copper centers. At best, a twofold increase of T1 from g perpendicular to g parallel was measured. Optimized fits of the saturation-recovery data were obtained using both double-exponential and stretched-exponential functions. The temperature dependence of the spin-lattice relaxation rate of mutant N2OR is about T5.0 with the stretched-exponential model or T3.3 and T3.9 for the model using the sum of two exponentials. These T1s are intrinsic to the mixed valence [CuA(1.5) ... CuA(1.5)] center, and no interaction of the second copper center in wild-type N2OR with the [CuA(1.5) ... CuA(1.5)] center has been observed. The T1 of the mixed valence center of N2OR is not only shorter than for monomeric square planar Cu(II) complexes, but also shorter than for a synthetic mixed valence complex, Cu2(N[CH2CH2NHCH2CH2NHCH2CH2]3N). The short T1 is attributed to the vibrational modes of type 1 copper and/or the metal-metal interaction in [CuA(1.5) ... CuA(1.5)].

New capabilities using saturation-recovery (SR) EPR at X-band (9.4 GHz) and W-band (94 GHz) to obtain profiles of the membrane fluidity have been demonstrated for dimyristoylphosphatidylcholine (DMPC) membranes with and without 50 mol% cholesterol. Phosphatidylcholine (n-PC) spin labels were used. Results were compared with profiles of the rotational diffusion coefficient, Rperp, obtained from simulation of EPR spectra using Freed's model. The spin-lattice relaxation rate (T1−1) obtained from SR EPR measurements of phospholipid spin labels in deoxygenated samples depends primarily on the rotational correlation time of the nitroxide moiety within the lipid bilayer. Thus, T1−1 can be used as a convenient quantitative measure of membrane fluidity that reflects membrane dynamics at a certain depth in the membrane. The order parameter, which is often used as a measure of membrane fluidity, describes the amplitude of wobbling motion of alkyl chains relative to the membrane normal and does not explicitly contain time or velocity. Thus, the order parameter can be considered as “nondynamic”. It is shown that T1−1 and Rperp profiles reveal changes in membrane fluidity that depend on the motional properties of the lipid alkyl chain. We find that cholesterol has a rigidifying effect only to the depth occupied by the rigid steroid ring structure and a fluidizing effect at deeper locations. These effects cannot be differentiated by profiles of the order parameter. Results demonstrate that SR EPR at W-band has the potential to be a powerful tool for studying samples of small volume, ∼30 nL, compared with a sample volume of ∼3 μL typically required at X-band.

This work demonstrates the use of multiquantum EPR to study the magnetic properties of copper complexes and copper proteins. Pure absorption spectra are obtained because of the absence of field modulation. The signal intensity of 3-quantum spectra is proportional to the spin lattice relaxation time T1, while its linewidth in a frequency difference sweep is T1(-1). A change in lineshape for the EPR detectable mixed value [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase is attributed to suppression of the forbidden transitions. The data confirm the unusually fast relaxation time for this site, which requires temperatures of less than 100 K to resolve hyperfine structure. The T1's for the mixed valence [Cu(1.5) . . . Cu(1.5)] site in nitrous oxide reductase are very similar to T1's for the Cua site in cytochrome c oxidase. The similar relaxation properties, together with previous multifrequency EPR results, support the hypothesis that the EPR detectable sites in cytochrome c oxidase and nitrous oxide reductase are mixed valence [Cu(1.5) . . . Cu(1.5)] configurations.

A double octarepeat prion protein construct, which has two histidines, mixed with copper sulfate in a 3:2 molar ratio provides at most three imidazole ligands to each copper ion to form a square-planar Cu(2+) complex. This work is concerned with identification of the fourth ligand. A new (to our knowledge) electron paramagnetic resonance method based on analysis of the intense features of the electron paramagnetic resonance spectrum in the g( perpendicular) region at 2 GHz is introduced to distinguish between three and four nitrogen ligands. The methodology was established by studies of a model system consisting of histidine imidazole ligation to Cu(2+). In this spectral region at 2 GHz (S-band), g-strain and broadening from the possible rhombic character of the Zeeman interaction are small. The most intense line is identified with the M(I) = +1/2 extra absorption peak. Spectral simulation demonstrated that this peak is insensitive to cupric A(x) and A(y) hyperfine interaction. The spectral region to the high-field side of this peak is uncluttered and suitable for analysis of nitrogen superhyperfine couplings to determine the number of nitrogens. The spectral region to the low-field side of the intense extra absorption peak in the g( perpendicular) part of the spectrum is sensitive to the rhombic distortion parameters A(x) and A(y). Application of the method to the prion protein system indicates that two species are present and that the dominant species contains four nitrogen ligands. A new loop-gap microwave resonator is described that contains approximately 1 mL of frozen sample.

Small uni-lamellar vesicles were prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with 1% spin label of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine (16-PC). POPC membranes are in the fluid phase in the physiological temperature range, which ensures high solubility of oxygen in the membrane center where the 16-PC nitroxide moiety is located. The nitroxide moiety is isolated from cellular reductants and paramagnetic ions that might interfere with spin-label oximetry measurements. This analyte is stable in cell suspensions and can be used in studies as long as 400 min. The saturation recovery EPR approach, possibly for the first time, was used to study cellular respiration of rat dopaminergic neuronal cells. It is widely accepted that this approach is the most direct way to carry out oximetric studies. The preliminary experiments were carried out at X-band for closed chamber geometry. Oximetric measurements were performed for cell concentrations from 750 to 5000 cells in one µL of culture media. Results confirmed that the oxygen consumption rate was linearly proportional to the number of cells. Furthermore at the lower cell concentration, we found a breaking point in the rate which is not due to diffusion limitation but is due to the change in cell metabolism because there is not enough oxygen, consistent with Michaelis-Menten kinetics. In the developed analyte, the same hydrocarbon environment, which dissolves oxygen very well, always surrounds nitroxide moieties of spin-label molecules. Therefore, the partial pressure of oxygen influence the EPR spectrum of spin-labels in the analyte. Such microscopic analytes are readily and uniformly distributed within the sample, thus giving a rapid response to changes in oxygen partial pressure. The improved oximetry methodology developed here presents a high impact opportunity for non-invasive assessment of disease mechanisms and response to therapeutic intervention.

The native state ensemble of a protein is comprised of distinct conformational substates in equilibrium, exchanging on the μs - ms time scale. Functionally important substates can be sparsely populated (“excited states”) and invisible to conventional spectroscopic methods, but recent studies have shown that high hydrostatic pressure can populate excited states and slow exchange kinetics to the easily measurable millisecond and longer time window. Thus, hydrostatic pressure can populate excited states for spectroscopic characterization, and pressure-jump relaxation experiments can determine the exchange rates between excited and ground states. Site Directed Spin Labeling (SDSL) - Electron Paramagnetic Resonance (EPR) spectroscopy of proteins containing a paramagnetic nitroxide side chain (a “spin label”) has been shown to provide information on both structure and dynamics in proteins of any degree of complexity. This makes it an ideally suited spectroscopic technique for these studies. Here we present a pressure-jump EPR system that achieves sub-millisecond pressure jumps or drops of arbitrary magnitude. We use a pressure intensifier capable of generating hydrostatic pressures up to 4 kbar (60,000 psi) to pressurize two reservoirs, and a novel air operated valve to rapidly equilibrate the reservoirs to an intermediate final pressure. Pressure cells and resonators have been developed for both X and Q band operation; the Q band system employs a modified Varian E-110 Q-band bridge. The pressure-jump system and EPR spectrometer are controlled via a user interface constructed with Labview. Together, these components comprise a complete pressure-jump EPR system for quantifying millisecond or slower conformational exchange rates in proteins. Preliminary results on conformational exchange rates in apo-myoglobin and T4 Lysozyme cavity mutants will be presented.

We have used saturation-recovery electron paramagnetic resonance (SR-EPR), a time-resolved saturation transfer EPR technique, to measure directly the microsecond rotational diffusion of spin-labeled proteins. SR-EPR uses an intense microwave pulse to saturate a spin population having narrow distribution of orientations with respect to the magnetic field. The time evolution of the signal is then observed. The signal increases in time as saturation is relieved by spin-lattice relaxation (Tl) as well as by saturation transfer due to spectral diffusion (Tsd), which is a function of rotational diffusion (Tr) and spectral position. In the presence of both events, the recovery is biphasic, with the initial phase related to both Tr and Tl, and the second phase determined only by Tl. We have measured the saturation recoveries of spin-labeled hemoglobin tumbling in media of known viscosities as a function of rotational correlation time (Tr) and pulse duration (tp). The Tr values estimated from the initial phase of recovery were in good agreement with theory. Variation of the pulse time can also be used to determine Tr. For tp less than Tsd, the recoveries were observed to be biphasic, for tp greater than Tsd a single-exponential. T1 values were determined from the recoveries after pulses quenching spectral diffusion or from the second phase of recovery after shorter pulses. These results demonstrate that SR-EPR is applicable to the study of motion of spin-labeled proteins. Its time resolution should provide a significant advantage over steady state techniques, particularly in the case of motional anisotropy or system heterogeneity.

Previously, saturation transfer (ST-EPR) studies of biomolecular dynamics have involved the use of a resonant cavity and the V'2 display (absorption, second harmonic, out of phase). In the present study, we replaced the resonant cavity with a loop-gap resonator and used the U'1 display (dispersion, first harmonic, out of phase) to study spin-labeled muscle fibers. The new resonator and display showed several advantages over those previously used. It produced virtually noiseless U'1 spectra on a 0.4 microliter sample using a 4 min scan; previous U'1 experiments on spin-labeled muscle, using a conventional rectangular cavity, resulted in an unacceptably low signal-to-noise ratio. The high filling factor of the resonator facilitated the study of these extremely small fiber bundles and permitted high microwave field intensities to be achieved at much lower incident microwave power levels, thus greatly enhancing the signal-to-noise ratio in U'1 experiments. This reduction in the noise level made it possible to benefit from the other advantages of U'1 over V'2, such as stronger signals, simpler line shapes, and simpler data analysis. For these muscle fiber samples, the resulting sensitivity (signal/noise/sample volume) of the U'1 signals was greater than 100 times that of V'2 signals obtained in a conventional cavity. Another advantage of the U'1 display is that signals from weakly immobilized probes, i.e., probes that have nanosecond rotational mobility relative to the labeled protein (myosin), are greatly suppressed relative to strongly immobilized probes. This reduces the ambiguity of spectral analysis, and eliminates the need for chemical treatments [e.g., using K3Fe(CN)6] that were previously required in muscle fibers and other systems. Further suppression of this weakly immobilized component was achieved in U'1 spectra by increasing the microwave power and decreasing the field modulation frequency.

Electron-electron double resonance (ELDOR) and saturation-recovery spectroscopy employing 14N:15N stearic acid spin-label pairs have been used to study the effects of cholesterol on lateral diffusion and vertical fluctuations in lipid bilayers. The 14N:15N continuous wave electron-electron double resonance (CW ELDOR) theory has been developed using rate equations based on the relaxation model. The collision frequency between 14N-16 doxyl stearate and 15N-16 doxyl stearate, WHex (16:16), is indicative of lateral diffusion of the spin probes, while the collision frequency between 14N-16 doxyl stearate and 15N-5 doxyl stearate, WHex (16:5), provides information on vertical fluctuations of the 14N-16 doxyl stearate spin probe toward the membrane surface. Our results show that: (a) cholesterol decreases the electron spin-lattice relaxation time Tle of 14N-16 doxyl stearate spin label in dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (egg PC). (b) Cholesterol increases the biomolecular collision frequency WHex (16:16) and decreases WHex (16:5), suggesting that incorporation of cholesterol significantly orders the part of the bilayer that it occupies and disorders the interior region of the bilayer. (c) Alkyl chain unsaturation of the host lipid moderates the effect of cholesterol on both vertical fluctuations and lateral diffusion of 14N-16 doxyl stearate. And (d), there are marked differences in the effects of cholesterol on lateral diffusion and vertical fluctuations between 0–30 mol% and 30–50 mol% of cholesterol that suggest an inhomogeneous distribution of cholesterol in the membrane.

The concentration of oxygen in the hydrocarbon region of lipid bilayer has been determined using a novel electron spin resonance (ESR) nitroxide-radical spin-probe method. For dimyristoylphosphatidylcholine (DMPC), the partition coefficient above the main transition temperature is approximately 3. Rapid decrease to 0.2 occurs below the pretransition temperature indicating exclusion of oxygen in the crystalline phase. The differences of molar free energy, enthalpy, and entropy of mixing between water and lipid have been determined for each phase.

Electron-nuclear double resonance (ENDOR) signals from matrix protons interacting with the stable free radicals of "A"- and "B"-type melanins have been observed as a function of pH. In all samples the single line is similar in width and unusually narrow. The ENDOR reduction varies by more than a factor of 10, indicating a large sensitivity of relaxation properties of melanin to sample type. Signals were observed over a wide range of experimental conditions with good signal-to-noise ratio, establishing feasibility for further more detailed ENDOR studies. Incubation in D2O resulted in little change, indicating that the free radical is well buried or protected. No resolved hyperfine structure was seen, consistent with the generally accepted view that melanin is a heterogeneous polymer.

The Smoluchowski equation for the bimolecular collision rate of dissolved oxygen molecules with spin labels yielded values for the diffusion constant of oxygen in water that are in agreement with the Stokes-Einstein equation (D infinity T/eta, where eta is the macroscopic viscosity) and with published values obtained by conventional methods. Heisenberg exchange at an interaction distance of 4.5 A occurs with a probability close to one for each encounter. In mixed hydrocarbons (olive oil, paraffin oils) and sec-butyl benzene, D infinity (T/eta)rho, where rho lies between 0.5 and 1. Oxygen diffuses in the hydrocarbons between 10 and 100 times more rapidly than predicted from the macroscopic viscosity. Similar results would be expected for diffusion of oxygen in model and biological membranes. Parallel measurements of rotational diffusion of the spin labels show little correlation with measurements of translational diffusion of oxygen. Dipolar interactions between spin labels and oxygen appear negligible except in the limit of highest viscosities.

Electron-electron double resonance (ELDOR) techniques employing [14N], [15N] 16-Doxylstearate spin-label pairs have been used to measure the lateral diffusion constant, D, of lipids in the surface membrane of intact human blood platelets. For freshly prepared platelets, D is 1.0 X 10(-8) cm2/s at 37 degrees C and for platelets stored for 3 d at room temperature under accepted routine blood bank conditions, D is 2.6 X 10(-8) cm2/s at 37 degrees C. This is the first time that D in the surface membrane of platelets is reported. The marked increase in D for stored platelets may be attributed at least partly to loss of cholesterol during storage, suggesting a correlation between lipid lateral diffusion and cholesterol levels in cell membranes.

Membranes made of dimyristoylphosphatidylcholine and cholesterol, one of the simplest paradigms for the study of liquid ordered-disordered phase separation, were investigated using a pulse-EPR spin-labeling method in which bimolecular collision of molecular oxygen with the nitroxide spin label is measured. This method allowed discrimination of liquid-ordered, liquid-disordered, and solid-ordered domains because the collision rates (OTP) differ in these domains. Furthermore, the oxygen transport parameter (OTP) profile across the bilayer provides unique information about the three-dimensional dynamic organization of the membrane domains. First, the OTP in the bilayer center in the liquid-ordered domain was comparable to that in the liquid-disordered domain without cholesterol, but the OTP near the membrane surface (up to carbon 9) was substantially smaller in the ordered domain, i.e., the cholesterol-based liquid-ordered domain is ordered only near the membrane surface, still retaining high levels of disorder in the bilayer center. This property may facilitate lateral mobility in ordered domains. Second, in the liquid-disordered domain, the domains with approximately 5 mol % cholesterol exhibited higher OTP than those without cholesterol, everywhere across the membrane. Third, the transmembrane OTP profile in the liquid-ordered domain that contained 50 mol % cholesterol dramatically differed from that which contained 27 mol % cholesterol.

Three- and five-quantum absorption and dispersion multiquantum electron paramagnetic resonance spectra of a spin-labeled protein have been obtained for the first time at Q-band (35 GHz). Spectra of arrestin spin-labeled at site 267 were recorded at room temperature as a function of microwave power. The separation of irradiating microwave frequencies, Deltaf, was 10 kHz, and a newly-designed multiquantum Q-band electron paramagnetic resonance bridge was utilized, operating in a superheterodyne detection mode. The sample volume was 30 nL using a 3-loop-2-gap resonator. Most spectra were obtained at a 300 microM concentration in single, 2-min scans, but spectra were also successfully obtained at 30 microM, corresponding to one picomole of protein. Enhanced sensitivity to T(1) and T(2) was evident in the spectra, and linewidths varied considerably across the spectra. The pure absorption displays are beneficial relative to field modulation methods for spectral characterization. The presence of two states of the nitroxide spin-label with different relaxation times is evident, particularly in the dispersion spectra, which are expected to exhibit enhanced sensitivity to lineshape variation relative to absorption. Feasibility has been established for the use of this technique for site-directed spin-labeling studies of biologically relevant samples, particularly the study of protein structure and dynamics.

Short-pulse saturation-recovery (SR) electron spin resonance (ESR) methods have been used to measure the lateral diffusion of a nitroxide-labeled cholesterol analogue (3-spiro-[2'-(N-oxyl-4',4'-dimethyloxazoladine)]-cholestane, CSL) in multilamellar liposomal dispersions. SR experiments were performed on samples containing 14NCSL:15NCSL pairs, and recovery signals were analyzed for initial conditions and multiexponential time constants by computer simulation. Rate equations describing the system were written and solved. The time constants contain combinations of electron spin lattice relaxation times Tle for both isotopes and the Heisenberg exchange rate constant Kx. We have investigated the complication that occurs from overlap of ESR spectral fragments from 14N and 15N moieties. The time constants of the multiexponential signals are independent of ESR line shape and position. From Kx, lateral diffusion constants of CSL in dimyristoylphosphatidylcholine (DMPC) were calculated (D = 1.7 x 10(-8) at 27 degrees C and 2.7 x 10(-8) cm2/s at 37 degrees C). It is shown that short-pulse saturation-recovery methods are able to overcome the ESR spectral overlap problem that is encountered in conventional ESR and continuous wave electron-electron double resonance (CW ELDOR) studies of spin-spin interactions. The present method can be extended to more complex situations involving spin labels in different environments with physical and chemical exchange.

To obtain correct EPR line shapes, spin-lattice relaxation times, and bimolecular collision rates between spin labels and oxygen, samples must be thoroughly deoxygenated or equilibrated with a controlled oxygen partial pressure. These measurements are conveniently carried out using a gas permeable plastic sample tube of small diameter that fits in a loop-gap resonator. Flow of gas over the tube allows easy deoxygenation or controlled oxygenation of the sample. In the initial design of the W-band (94 GHz) loop-gap resonator, samples were equilibrated with gas at room temperature outside the resonator, transferred to a quartz capillary, and positioned in the resonator. In a recently designed W-band loop-gap resonator, the sample is positioned in a gas-permeable Teflon tubing inside the resonator, which allows measurements of extremely small volume (∼30 nL) of sample. We used this new design to measure properties of lens lipid membranes derived from total lipids extracted from both lenses (single donor) of a 2-year-old porcine cortex and nucleus. Detailed profiles of membrane fluidity and oxygen transport parameter were obtained from saturation recovery EPR. Analysis of conventional spectra using the microscopic-order macroscopic-disorder (MOMD) model provided rotational diffusion coefficients (R(perpendicular) and R(parallel)) and order parameters. Three different types of motion of lipid spin labels n-PC, T-PC, and CSL (ASL) with, respectively, nitroxide z-axis, x-axis, and y-axis parallel to the bilayer normal, are discussed. Results demonstrate that EPR at W-band has the potential to be a powerful tool for studying samples of small volume, ∼30 nL, obtained from eye lenses of a single human donor.